Mobilizable plasmids drive the spread of antimicrobial resistance genes and virulence genes in Klebsiella pneumoniae

Background Klebsiella pneumoniae is a notorious clinical pathogen and frequently carries various plasmids, which are the main carriers of antimicrobial resistance and virulence genes. In comparison to self-transmissible conjugative plasmids, mobilizable plasmids have received much less attention due to their defects in conjugative elements. However, the contribution of mobilizable plasmids to the horizontal transfer of antimicrobial resistance genes and virulence genes of K. pneumoniae remains unclear. In this study, the transfer, stability, and cargo genes of the mobilizable plasmids of K. pneumoniae were examined via genetic experiments and genomic analysis. Methods Carbapenem-resistant (CR) plasmid pHSKP2 and multidrug-resistant (MDR) plasmid pHSKP3 of K. pneumoniae HS11286, virulence plasmid pRJF293 of K. pneumoniae RJF293 were employed in conjugation assays to assess the transfer ability of mobilizable plasmids. Mimic mobilizable plasmids and genetically modified plasmids were constructed to confirm the cotransfer models. The plasmid morphology was evaluated through XbaI and S1 nuclease pulsed-field gel electrophoresis and/or complete genome sequencing. Mobilizable plasmid stability in transconjugants was analyzed via serial passage culture. In addition, in silico genome analysis of 3923 plasmids of 1194 completely sequenced K. pneumoniae was performed to investigate the distribution of the conjugative elements, the cargo genes, and the targets of the CRISPR-Cas system. The mobilizable MDR plasmid and virulence plasmid of K. pneumoniae were investigated, which carry oriT but lack other conjugative elements. Results Our results showed that mobilizable MDR and virulence plasmids carrying oriT but lacking the relaxase gene were able to cotransfer with a helper conjugative CR plasmid across various Klebsiella and Escherichia coli strains. The transfer and stability of mobilizable plasmids rather than conjugative plasmids were not interfered with by the CRISPR–Cas system of recipient strains. According to the in silico analysis, the mobilizable plasmids carry about twenty percent of acquired antimicrobial resistance genes and more than seventy-five percent of virulence genes in K. pneumoniae. Conclusions Our work observed that a mobilizable MDR or virulence plasmid that carries oriT but lacks the relaxase genes transferred with the helper CR conjugative plasmid and mobilizable plasmids escaped from CRISPR–Cas defence and remained stable in recipients. These results highlight the threats of mobilizable plasmids as vital vehicles in the dissemination of antibiotic resistance and virulence genes in K. pneumoniae. Supplementary Information The online version contains supplementary material available at 10.1186/s13073-023-01260-w.


Supplementary figures
Fig. S1.The mobilizable MDR plasmid pKPHS3 can be transferred between different K. pneumoniae strains with the help of CR plasmid pKPHS2.The recipient strains are RJF999 and RJF293H.Detailed data are available in Additional file 6: Table S5.(c) XbaI PFGE and S1-PFGE of transconjugants and their parental strains.M represents the molecular weight marker, Salmonella serotype Braenderup H9812 strain.Strains with the same symbol on the PFGE image represent the progeny derived from the same parental strain.The purple triangle denotes the location of the band of pKPHS3 and the strain harboring pKPHS3 is also validated by PCR.The number of plasmids corresponds to the size of the pie charts.pneumonia RJF293, and its transconjugants including RJF293-p2 and RJF293-p2-p3.

Fig. S2 .
Fig. S2.The mobilizable virulence plasmid pRJF293 can be transferred between different Klebsiella strains with the help of CR plasmid pKPHS2.

Fig. S3 .
Fig. S3.MDR plasmid pKPHS3 and virulence plasmid pRJF293 can be transferred into E. coli C600 in various patterns with the help of CR plasmid pKPHS2.

Fig. S7
Fig.S7The mobilizable virulence plasmid pRJF293 and the mobilizable MDR plasmid pKPHS3 can be transferred into KpBSI083A and its derivate KpBSI083Δcas3H with the help of conjugative CR plasmid pKPHS2 (p2).

Fig. S8 .
Fig. S8.Proposed mechanism of KpBSI083A-p3 and KpBSI083A-pV generation.Fig. S9.Distribution of CRISPR-Cas systems and plasmids in 1,194 completely sequenced K. pneumoniae strains taken from GenBank.Fig.S10.The number of plasmids targeted and nontargeted by endogenous CRISPR-Cas systems.Fig.S11.Distribution of conjugative, mobilizable, and nonmobilizable plasmids according to plasmid size in six typical STs of K. pneumoniae strains.Fig.S12.Distribution and mobility of clinically alarming plasmids in K. pneumoniae.

Fig. S1 .
Fig. S1.The mobilizable MDR plasmid pKPHS3 (p3) can be transferred between different K. pneumoniae strains with the help of CR plasmid pKPHS2 (p2).(a) Schematic diagram of the conjugation assays.The green square denotes the tra gene cluster on pKPHS2.The green square with white slashes denotes the incomplete tra gene cluster on pKPHS3.(b) The conjugation frequencies of pKPHS2 and pKPHS3.The donor strains are HS11286, RJF999-p2 and RJF999-p2-p3.

Fig. S2 .
Fig. S2.The mobilizable virulence plasmid pRJF293 (pV) can be transferred between different Klebsiella strains with the help of CR plasmid pKPHS2 (p2).(a) Schematic diagram of the conjugation assays.The green square denotes the tra gene cluster on pKPHS2.The green square with white slashes denotes the incomplete tra gene cluster on pKPHS3 (p3).(b) The conjugation frequencies of pKPHS2 and pRJF293.The donor strains are RJF999-p2, RJF293-p2 and KvBSI002A-p2-pV.The recipient strains are KvBSI002A and HS11286.Detailed data are available in Additional file 6: Table S5.(c) XbaI PFGE and S1-PFGE of transconjugants and their parental strains.M represents the molecular weight marker, Salmonella serotype Braenderup H9812 strain.Strains with the same symbol on the PFGE image represent the progeny derived from the same parental strain.The purple triangle denotes the location of the band of pKPHS3 and the strain harboring plasmid pV is also validated by PCR.

Fig. S4 .
Fig. S4.Genetic structure of the fusion p2-3 of the transconjugant E. coli C600-p2-3.The tra genes are indicated in green, IS elements are shown in black and resistance genes are shown in fuchsia.The hybrid p2-3 has a length of 196,720 bp and a GC content of 52%.

Fig. S5 .
Fig. S5.Genetic structure of the fusion plasmids p2-V-1 and p2-V-2 of the transconjugant E. coli C600-p2-V.(a) The p2-V has a length of 264,857 bp and a CG content of 51% and contains tra genes and virulence genes.(b) The p2-V-2 has a length of 74,014 bp and a CG content of 54% and contains the carbapenemase gene blaKPC.The tra genes are indicated in green.Virulence genes are indicated in red.IS elements are shown in black.Resistance genes are shown in fuchsia.(c) The speculative process of formation of p2-V and p2-V-2.The red circle presents the backbone of plasmid pV and the blue circle presents the backbone of pKPHS2 (p2).The black band is the region of ISpa40.

Fig. S7
Fig. S7 The mobilizable virulence plasmid pRJF293 (pV) and the mobilizable MDR plasmid pKPHS3 (p3) can be transferred into KpBSI083A and its derivate KpBSI083Δcas3H with the help of conjugative CR plasmid pKPHS2 (p2).(a) Schematic diagram of the conjugation assays.The green square denotes the tra gene cluster on pKPHS2.The green square with white slashes denotes the incomplete tra gene cluster on pKPHS3.(a) XbaI PFGE and S1-PFGE of transconjugants and their parental strains.M represents the molecular weight marker, Salmonella serotype Braenderup H9812 strain.Strains with the same symbol on the PFGE image represent the progeny derived from the same parental strain.

Fig. S9 .
Fig. S9.Distribution of CRISPR-Cas systems and plasmids in 1,194 completely sequenced K. pneumoniae strains taken from GenBank.The left bar chart shows the proportion of K. pneumoniae strains in the presence of the CRISPR -Cas system.The green bar indicates the K. pneumoniae strains containing CRISPR-Cas systems.The middle heatmap shows the distribution of plasmids with different replicons in the K. pneumoniae strains of the top 20 STs associated with the highest number of plasmids.The right bar chart presents the proportion of plasmids with different transferable abilities.The top bar chart shows the number of resistance genes and acquired virulence genes per plasmid with different replicons.The bottom bar chart indicates the scale of plasmid mobility in plasmids with different replicons.

Fig. S10 .
Fig. S10.The number of plasmids targeted and nontargeted by endogenous CRISPR -Cas systems.From left to right, the first five columns represent the plasmids from the cKP strains (ST11, ST258, ST307, ST231, and ST37) without CRISPR -Cas system are targeted by CRISPR -Cas systems of K. pneumoniae from strains (ST14, ST15, ST23, ST45, and ST147).The sixth column represents the plasmids in those strains targeted by all CRISPR-Cas systems in K. pneumoniae.The last column represents the plasmids without targets of the CRISPR -Cas system in K. pneumoniae.

Fig. S11 .
Fig. S11.Distribution of conjugative, mobilizable, and nonmobilizable plasmids according to plasmid size in six typical Sequence Types of K. pneumoniae strains.Six typical STs ranging from ST11 to ST15 were selected to investigate the density and distribution of plasmids.The medians of plasmid size of conjugative plasmid, mobilizable plasmid, and nonmobilizable plasmid were presented via blue, pink, and gray dotted lines, respectively.

Fig. S12 .
Fig. S12.Distribution of clinically alarming plasmids in K. pneumoniae.(a) MDR plasmids carried by different MLST host strains.(b) Mobilities of MDR plasmids.(c) CR plasmids carried by different MLST host strains.(d) Mobilities of CR plasmids.(e) Virulence plasmids carried by different MLST host strains.(f) Mobilities of virulence plasmids.The proportion of mobilizable plasmids that co-occurred with (blue) or without (gray) conjugative plasmids was indicated via small pie charts.

Fig. S14 .
Fig. S14.Proposed transfer interaction of the mobilizable plasmid and conjugative plasmid.The conjugative plasmids encode four modules for plasmid conjugation, including the relaxase which could recognize and nick oriT on plasmid.The mobilizable plasmids carrying both an oriT and a relaxase gene (pMOB) can transfer with the help of conjugative elements encoded by conjugative plasmids.The mobilizable plasmids carrying an oriT but lacking a relaxase gene (pOriT) could transfer under the help of relaxase encoded by other plasmids and the aid of conjugative elements supported by the conjugative plasmid.